In December Eric Betzig is expected to fly to Stockholm to receive his share of the 2014 Nobel Prize in Chemistry for expanding the frontiers of microscopy. It seems he just couldn’t leave those frontiers alone.

In a tour de forcepaper in Science, Betzig and his collaborators have introduced a new method for imaging biological processes with unprecedented resolution in space and time. Betzig and Bi-Chang Chen, Wesley R. Legant, and Kai Wang from his lab at the Howard Hughes Medical Institute’s Janelia Research Campus were joined by collaborators from 14 other groups around the world to come up with the new microscopy method.

The technique, called lattice light-sheet microscopy, generates extraordinarily sharp, 3-D images and videos of live organisms at scales ranging from single molecules to early-stage embryos. It builds on other methods Betzig has pioneered.

One of those methods was something the Nobel Prize committee mentioned in making their award, Betzig’s development of photoactivated localization microscopy (PALM). PALM lets researchers see objects smaller than the half-wavelength diffraction limit—by shining less light on the subject, rather than more.

The diffraction limit is the light microscopist’s nemesis. Violet light has the shortest wavelengths most humans can see, down to about 380 nanometers (though some people who have lost their corneas to cataract surgery can see into the ultraviolet, to perhaps 300 nanometers). Thus, objects smaller than about 200 nanometers are invisible to conventional light microscopy. In PALM, Betzig linked fluorescent molecules to proteins in the feature he wanted to study, and then shined a light to stimulate them. The labeled molecules responded weakly, with only a small, widely separated percentage of the fluorescent molecules emitting a few photons. When the emitting molecules were farther than 200 nm apart, each produced a single, highly localized bright spot on the image. Betzig then stimulated and imaged the sample over and over again, hundreds of times, to build a mosaic of sub-200 nm detail.

The new lattice light-sheet technique is also based on a Bessel beams method the Janelia group introduced in 2011. Bessel beams are nondiffracting wave patterns, vanishingly-thin rings of light produced by shining a laser through an annular mask. These beams keep their shape, and don’t spread out as they propagate, allowing researchers to generate extremely thin sheets of light.

Image: Betzig Lab/HHMI
Lattice light-sheet microscopy allows the tracking of single molecules of the protein that marks the ends of growing microtubules.

In their new paper, Betzig’s team says that they have “crafted ultrathin light sheets.” Crafted is the right word: they shape the light the way a carpenter turns, shaves, and carves a baulk of lumber into a graceful table leg.

First, the laser beam is squeezed and stretched into a thin vertical line. Then it is bounced off a spatial light modulator (SLM)—an ultra-fast LCD that flashes a series of black-and-white patterns that reflect the incoming stripe of laser light back in a defined lattice of high and low intensity. (The SLM is the main feature distinguishing the lattice sheet method from its Bessel beam predecessor.) The reflected pattern passes through a thin transform lens, which throws a Fourier transform of the pattern onto the annular Bessel beam mask. The light then bounces between galvanometer-controlled mirrors that determine where the nodes of the lattice will fall in the light sheet. Only then does the beam pass through a lens to be focused into the sample, producing a hexagonal pattern of illuminated dots that spread in a sheet right and left of the beam. If the pattern is chosen wisely, interference effects actually sharpen the definition of the points, increasing resolution.

A piezoelectric stage moves the sample incrementally through the light sheet, which activates fluorescent markers in an ultrathin slice of the sample.

The lattice light-sheet device works in two modes—structured illumination microscopy (SIM) for high spatial resolution and “dithered” for better time resolution. SIM can resolve features in the 150-280 nm range. In the high-speed dithering mode, the lattice is swept across the sheet faster than the camera’s exposure time, illuminating a whole slice in a single step to reduce the number of steps necessary to photograph an entire volume. Dithered mode functions at 100 frames per second—about 7.5 times faster than SIM mode but, at at 230-370 nm, it only has two-thirds SIM’s resolution.

No matter how kindly intended, a flood of photons pouring into a cell can injure or kill it—especially if it is dividing or otherwise vulnerable. The lattice light-sheet technique doesn’t do that. “The chief benefit of lattice light-sheet excitation… is its exceptionally low photobleaching and phototoxicity,” the researchers write. Each of the examples presented in the paper “was distilled from tens or hundreds of thousands of raw 2-D images,” a figure one or even two magnitudes higher than the number of exposures allowable with other light methods.

The research produced remarkable images from a large collection of experiments conducted in collaboration with colleagues from 14 other groups around the world. Here are just a few of them:

Repeatedly imaging a non-living fixed sample to localize 4.2 million individual proteins in the nucleus’ membrane to within 8 to 45 nm.

Tracking single molecules of the protein that marks the ends of growing microtubules, the fibers that latch onto and reposition the replicating chromosomes during cell division, to build a comprehensive dynamic map of their growth [image above].

Keeping the light sheet stationary to follow extremely rapid changes in a protozoan at more than 300 frames per second, including stop-action images of moving cilia that could allow biologists to calculate the force each of the tiny tails generates.

Filming details of key stages in the development of live fruit fly embryos in unprecedented detail.

The researchers have patented the lattice light-sheet microscopy system. They’ve licensed it to microscope maker Zeiss and will also share the detailed instructions to researchers who wish to build their own instruments.

Where many scientific abstracts close with a guarded boast about future potential, this one ends: “The results provide a visceral reminder of the beauty and complexity of living systems.”

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Rapid muscle contractions in a C. elegans embryo in the three-fold stage, with labeled GFP-PH domains (green) and mCherry-histones (magenta), as recorded in a single 2D optical section at 50 frames/sec. Scale bar, 10 um. (Video: Betzig Lab/HHMI)

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